Pullulanase (EC 3.2.1.41) is one of the starch-debranching enzymes which is widely used in lots of areas such as food and pharmaceutical industries. It is necessary to explore pullulanases with high activities and good properties for cost cuttings of agricultural product's deep processing and starch industries. In previous work, an efficient pullulan-degrading bacterium Klebsiella variicola strain Z-13 had been isolated from the soil near a starch factory. In this study, the pullulanase from the fermentation broth was purified by 80% ammonium sulphate precipitation, Sephadex G-25 and Sephadex G-100 chromatography. The pure enzyme's activity was 51.15 U/mg and the recovery rate was 26.9%. SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the molecular weight of this protein was 120 kD. The enzymatic properties were also studied. Thin layer chromatography (TLC) analysis showed that the enzymatic product of pullulan was maltotriose. The optimal temperature of the enzyme was 45 ℃ and the optimal pH was 5.6. The pure pullulanase was very stable at both optimal temperature (45 ℃) and optimal pH (pH5.6), and 80% activity still remained even after 120 min at 50 ℃. Zn2+, Na+ and Ca2+ promoted the enzyme's activity at the final concentration of 5 mmoL/L while K+, Mg2+, Al3+ and Co2+ inhibited the enzyme's activity at the same concentration. The activity was completely lost when the enzyme was incubated with Cu2+. Moreover, according to the pullulanase gene published on NCBI from Klebsiella variicola SHN-1 (Accession No. JX087429.1), the primers were designed and the pullulanase gene of Klebsiella variicola strain Z-13 was successfully cloned (Accession No. KJ146839). The similaritiy of the two genes reaches 98.88% while the amino acids sequence similarity was 98.18%. In addition, the amino acids sequence had a highly conserved region YNWGYDP of typeⅠpullulanase at the N terminal, and also 4 conserved domains of GH 13 family. This research purified a typeⅠpullulanase from Klebsiella variicola with relative good activity and stability, which has potential industrial uses. The cloning of the pullulanase gene of Klebsiella variicola strain Z-13 provides the possibility for further studies such as heterogeneous expression and molecular modification of the enzyme.

Plant defensins are kinds of small molecular proteins with typical three-dimensional structures, which are composed of cysteine residues, and are also one of important active substances in the process of plant defense. In this study, based on tomato (Solanum lycopersicum) whole genome sequencing data, identification, location, structure, phylogenetic relationship and expression patterns of the tomato defensin gene were performed using bioinformatics methods. Results showed that the tomato genome contained 15 defensin genes, the amino acid sequence length of which was 73~105 aa, while the molecular weight was 8.09 to 11.91 kD. Sequence alignment found that all these members contain Gamma-thionin conservative domain, which was characteristic of defensin genes. Chromosomal location showed that these genes were mapped on 4 chromosomes (Chr 04 Chr 07 Chr 09 and Chr 11). Phylogenetic relationship revealed that the tomato and Arabidopsis thaliana defensin genes could be divided into 7 groups, only one pair was orthologous gene. RNA-Seq analysis found that 6 defensin genes showed tissue-specific expression, expressions of 2 defensin genes were broad spectrum of patterns, and 5 were low in all tissues tested. qRT-PCR results showed that only 2 defensin genes (Solyc07g007750 and Solyc07g007760) were induced by tomato yellow leaf curl virus. All these results suggested that the defensin genes not only involve in the tomato vegetative and reproductive growth, but also may participate in tomato responsing to biotic stresses. This work provides basic data for further function study of the defensin gene family in tomato.

Virus-induced gene silencing (VIGS) has been widely used in plant gene functional analysis. In the present study, in order to obtain VIGS candidate Cucumber mosaic virus CMV strain, (CMV) pseudo-recombinants between RNA2 from different CMV strains and CMV Fny strain (CMV-Fny) RNA1 and RNA3 were researched. Results of pseudo-recombinants CMV infection indicated that CMV -F1Tsh2F3 consisting of CMV-Fny genomic RNA1, RNA3 and RNA2 of CMV-TshR2 induced slightly mosaic symptom on tabacco (Nicotiana benthamiana), while the other CMV pseudo-recombinants caused host plants to produce stunt or leaf curf at 7 days post-inoculation(dpi). In order to obtain the Agrobacterium-mediated CMV infectious cDNA clones, genomic RNA1~3 of CMV was cloned into binary expression vector pCB301. Agro-infiltration results of CMV infectious clones under the control of duplicated Cauliflower mosaic virus 35S promoter indicated that the pCB301-CMV phenotype on the host plants was identical to that induced by CMV in vitro trancripts RNAs. The seedlings of N. bethamiana could be infected by pCB301-F1Tsh2VIGSF3 systematically and expressed slightly mosaic symptom at 7 dpi, even though F1Tsh2VIGSF3 lacking 3' end 95 nucleotides of 2b gene. Western blot results indicated that coat protein (CP) of pCB301-F1Tsh2VIGSF3 was lower than that of pCB301-F1Tsh2F3. Chlorosis and yellowing symptom appeared on the whole N. benthamiana plants infected by pCB301-F1TshR2VIGS-Su351F3 at 10 dpi, and qRT-PCR analysis showed that the magnesium chelatase subunit gene of Sulphur mRNA levle of host plants was reduced by more than 80% compared with the CMV-infected control. Chlorosis and yellowing symptom on N. benthamiana induced by inoculation of 3 or 5 passages F1TshR2VIGS-Su351F3 was the same as that caused by infection with pCB301-F1TshR2VIGS-Su351F3, and so CMV-F1TshR2VIGS-Su351F3 existed stably in the host plants. In order to confirm whether CMV-VIGS could knock down other plant functional genes, the seedlings of N.tabacum cv. Xanthi NN was infiltrated with pCB301-F1TshR2VIGS-Beclinl337F3. The data suggested that the upper leaves on autophagy related gene of Beclin1-silenced hosts infected by Tobacco mosaic virus(TMV) produced necrosis symptom, and the Beclin1 mRNA levle of hosts infected by pCB301-F1TshR2VIGS-Beclinl337F3 was reduced by 70% compared with the control plants. Agrobacterium-mediated infectious cDNA clones of CMV and its VIGS vector supply a foundation for constructing CMV-VIGS vector in other host plants.

Growth hormone (GH) screened by the pituitary, is a polypeptide hormone which promotes the growth of the animal body, muscle development, and regulates metabolism and other important physiological functions. In this study, a pair of Mus musculus GH (mGH) short hairpin RNA(shRNA) was designed based on the 340bp-abasic site of mGH mRNA sequence, and the mGH shRNA sequence was linked to a integrated tetracycline induced expression vector with sequences syntheses method, the recombinant vector named as pSingle-tTS-mGH shRNA-RFP(red fluorescent protein). The recombinant vector was transfected to mice pituitary tumor cell lines AtT-20 and screened with G418 two weeks to enrich cell clones which integration of exogenous genes. The RFP expression was observed under a fluorescent microscope and the GH expression levels were detected with qRT-PCR and Western blot methods. The results showed, little RFP signal could be observed in control cell groups (cell groups transfected with pSingle-tTS-RFP plasmid) and experimental cell groups (cell groups transfected with pSingle-tTS-mGH shRNA-RFP plasmid) induced with doxycyclin (DOX), the RFP signal was obviously enhance in the above 2 cell groups. The mGH mRNA in control cell groups, control cell groups adding DOX, experimental groups and experimental groups induced with DOX was 1±0.17, 1.03±0.12, 0.758±0.19 and 0.204±0.07, respectively. Compared with the other 3 groups, the GH mRNA expression in experimental groups induced with DOX (working concentration, 600 ng/μL) difference between them was extremely obvious(P＜0.01), whereas the difference among the other 3 groups was not obvious (P＞0.05). The average GH protein expression level in control cell groups, control cell groups adding DOX, experimental groups and experimental groups induced with DOX was 1, 1.07, 0.88 and 0.32, respectively. An effective mGH shRNA sequence was designed in this study and a controllable mGH shRNA overexpression vector was constructed successfully and it showed well-controlled gene expression efficiency. The GH shRNA controllable expression system prepared in this study is a good tool to study the GH autocrine / paracrine interactions, function on body development and related disease through increase and decrease the GH expression in vitro and in vivo.

Baicalin is a kind of flavonoid, can promotes posttransplant fetal growth and development, and hasn't toxicity to the fetus development. The objectives of the present study were to investigate the effects of baicalin on the development of mouse (Mus musculus) embryos in vitro and it's possible molecular mechanism. Mouse embryos cultured in vitro were randomly divided into control group(CZB cultural medium) and experimental group (supplemented with 4 μg/mL of baicalin). The development rates of embryos in their different stages were recorded, respectively, using a stereoscopic microscope, and also the nuclei of blastocysts were stained with orcein for cell number counting. The expressions of gap junction protein alpha-1(GJA1), heat Shock 70 (Hsp70), B-cellymphoma/leukemia-2(Bcl-2) and Bcl-2 associated X protein (Bax) mRNA were detected by using qRT-PCR. The results showed that baicalin treatment, as compared to control group, significantly improved (P＜0.05) 2-cell and 4-cell embryonic cleavage rates, morula, blastocyst and expanded blastocyst development rates and blastocyst hatching rates, and also significantly (P＜0.05) improved the cell number of expanded and hatched blastocysts. In addition, the expression of GJA1 mRNA was significantly increased (P＜0.05), and the expressions of Hsp70 and Bax mRNA were significantly decreased (P＜0.05), and also the ratio between Bax/Bcl-2 was significantly reduced (P＜0.05). We concluded that baicalin improves the blastocyst development rates and the quality of blastocysts of mouse embryo in vitro, probably, by upregulating GJA1 mRNA expression and downregulating Hsp70 and Bax mRNA expressions. The study reveals the possible mechanism of action that the baicalin promotes embryonic development, and provides basic data for the study of baicalin tocolysis role.

Clotting disorders by molecular mismatch is one of the remaining critical issues in pig-to-human xenotransplantation. Thrombomodulin (TBM) is an endothelial transmembrane glycoprotein that plays a critical role in the control of coagulation. Studies demonstrated that pig (Sus scrofa) TBM can't effectively regulate blood clotting and thrombosis if the molecules do not match. Thus, the expression of exogenous human TBM in pig vascular endothelial cells may be helpful for overcoming the molecular incompatibilities. In this study, the 7 and 9 kb promoter of pig TBM gene were obtained by gap-repair method, and correspondingly 2 espression vectors of hTBM (p7K(pTBM)-hTBM, p9K(pTBM)-hTBM) were constructed. To validate and compare the specificity and effciency of the 2 vectors, both of them were transfected into endothelial cells, ear fibroblast and kidney cells of Wuzhishan miniature pig. The transfected cells were cultured for 48 h and then harvested for RT-PCR and Western blot analysis. The results of RT-PCR indicated that both the 7 and 9 kb promoters could specifically regulate the expression of hTBM in vascular endothelium; qRT-PCR and Western blotting analysis showed that the activity of 9 kb promoter was higher than the that of 7 kb counterpart (P＜0.05). Altogether, the results in vitro demonstrated that the 9 kb TBM promoter of pig was a more suitable candidate for endothelial cell specific expression, based on the fact that it could drive the expression of hTBM at higher efficiency . This work lays the foundation for the future transgenic pig efficiently express anti-coagulation factor in its vascular endothelium, which will solve the clotting disorders caused by a molecular mismatch in xenotransplantation.

As the hydrolysis of xylan is an important step towards the utilization of hemicelluloses, xylanases are widely used in the pulp and paper, animal feed, and brewing industries. In this work, amino acid alignment of xylanase from Bacillus subtilis with acidophilic and acid-stable xylanase from glycosyl hydrolase family 11 was carried by the ClustalW program. Then the mutants of N63D, A143E and N63D/A143E of xylanase were constructed by oligonucleotide-based site-directed mutagenesis methods, using the recombinant plasmid pUC18-xyl11 as the template. The mutated genes were inserted into expression vector pGEX-6p-1. And then, the recombinant plasmids pGEX-6p-xyl11, pGEX-6p-xyl11N63D, pGEX-6p-xyl11A143E and pGEX-6p-xyl11N63D/A143E were transformed into Escherichia coli BL21 (DE3) for protein expression and purification. After induction with IPTG(isopropyl β-D-thiogalactoside), the recombinant enzymes were successfully expressed in soluble form. The wild type Xyl11 and three mutants were purified to high homogeneity by glutathione-affinity chromatography followed by 3C protease digestion. SDS-PAGE analysis showed that the molecular mass of Xyl11 was about 20 kD. Then kinetic parameters of Xyl11 and mutants were determined. The optimum pH of the purified enzyme was determined within a pH range of 3.0~8.5. Xyl11 displayed the maximum activity at pH 6.0, while single site mutant, i.e., Xyl11N63D and Xyl11A143E, with showed the maximum activity at pH 4.5 and pH 5.0, respectively. Like Xyl11A143E, the pH optimum was 5.0. In comparison with the wild type enzyme, all the mutants shifted the pH optimum towards acid pH. The kinetic parameters of the purified xylanase and its mutants were determined by measuring the enzymatic activity with various concentrations of beechwood xylan as the substrate. The Michaelis constant(Km), Maximum reaction rate(Vmax) and catalytic number(Kcat)of Xyl11 were 5.996 mg/mL, 360.615 μmol/(min.mg) and 1 054.429 /s, respectively. The Km, Vmax and Kcat of Xyl11N63D was 6.876 mg/mL, 326.289 μmol/(min.mg) and 15.013 /s, respectively. The Km, Vmax and Kcat of Xyl11A143E was 9.178 mg/mL, 212.959 μmol/(min.mg) and 6.544 /s, respectively. For the mutant Xyl11N63D/A143E, Km was 14.885 mg/mL, Vmax was 201.248 (min.mg) and Kcat was 4.742 /s. According to these data, the Km of the mutans were increased, however, the Vmax and Kcat were decreased, compared to the wild type enzyme. Totally, it could be concluded that the sites Asn63 and Ala143 are involved in the pH optimum and activity of xylanase from Bacillus subtilis. This work provides insight into the structure-function study of xylanases, and valuable hints for the protein engineering of xylanases in future study.

To analyze the cytological behavior of synaptonemal complex (SC) related protein ZYP1 in plant meiosis, a recombinant vector pET28a-ZYP1 by cloning fragment of ZYP1 gene was constructed. The induction condition of the fusion protein expression in Escherichia coli was optimized. Polyclonal antibody was produced by immuning New Zealand rabbits (Oryctolagus cuniculus) with the protein which was purified by Ni-NTA affinity chromatography. The specificity and titer of the purified polyclonal antibody was examined by Western blot and ELISA. The results showed that the Arabidopsis thaliana ZYP1 prokaryotic expression vector was successfully constructed, and the optimized protein expression induction condition in E. coli was 1.0 mmol/L isopropy-β-D-thiogalactoside (IPTG) at 37 ℃ for 4 h. The polyclonal antibody had high specificity and sensitivity. It was able to detect and locate the antegin of ZYP1 protein in plant cells by immunofluorescence staining. The results of immunofluorescence staining showed that ZYP1 protein appeared at leptotene and gradually reduced at diplonema and nearly disappeared at diakinesis. The ZYP1 protein was an important part of regulating the synapsis of homologous chromosomes in meiosis, and the dynamic changes was consistent with the formation and dissolution of the synaptonemal complex. This study is helpful to deep analysis of the biological functions of ZYP1 in plant meiosis, which is the related protein of synaptonemal complex.

In order to investigate the molecular mechanism of salt tolerance, the transcriptomes of southern type Alfalfa (Medicago sativa ‘Millenium’) under control condition (WT_CK2) were compared with those under NaCl-treated (WT_CK2) condition. Total RNA was extracted from leaves under control and NaCl-treated conditions, and then was used for RNA Sequencing (RNA-Seq) analysis on the Illumina Hiseq 2000 platform. Meanwhile, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted on the differentially expressed genes (DEGs). qRT-PCR technique was used to validate the expression data of 6 randomly selected DEGs. The results indicated that after filtration of low-quality reads, a total of 62 771 040 and 60 394 756 high-quality reads were acquired, respectively, in control and NaCl-treated samples for the identification of DEGs. Among these high-quality reads, 52.47% (control) and 53.07% (NaCl-treated) could be accurately mapped to the reference genome of Medicago truncatula, thus validating the RNA-Seq data and the reference sequence. A total of 7 497 DEGs were identified, among which 4 078 and 3 419 were detected to be up-and down-regulated, respectively, under NaCl treatment. These genes include 474 transcription factors (TFs) from 46 TF families. GO enrichment analysis showed these DEGs were involved in pathways like binding, catalytic activity, cellular process, metabolic process and response to stimulus. KEGG pathway analysis showed that these DEGs were involved in biosynthesis of secondary metabolites, metabolic pathways, phenylpropanoid biosynthesis, favonoid biosynthesis and plant hormone signal transduction. Important DEGs contained genes involved in osmotic adjustment, ion transport, and pathways involved in biosynthesis and signal transduction. Genes coding calcium-dependent protein kinase, mitogen-activated protein kinase, PP2C family, ABA pathway and hormones (salicylic acid, ethylene and jasmonic acid) signal pathways were up-regulated, TFs from C3H, NAC, WRKY and AP2/EREBP TF families were also differentially up-regulated. On the other hand, genes related to fundamental metabolic mechanism and protein synthesis were generally down-regulated under salt stress. In addition, we identified many candidate genes, such as trehalose-6-phosphate synthase gene, vacuolar Na+/H+ antiporter gene, stress-induced protein kinase gene, calcium-dependent protein kinase gene, sucrose non-fermenting 1-related protein kinase, SnRK, germin-like protein gene, to be related to salt tolerance. Expression analysis of the 6 DEGs with qRT-PCR was in consistent with those of RNA-Seq data, thus furtherly confirming the reliability of RNA-Seq results. This research provides the basic evidence for the molecular mechanisms of salt tolerance in southern type alfalfa.

In order to understand the biological function of APETALA1 gene (AP1) in Paeonia lactiflora Pall., the present study designed primer based on conserved domain of higher plant AP1 and RACE (rapid-amplification of cDNA ends) cloned AP1 (GenBank No. KC354376) from Paeonia lactiflora. The molecular structure and function of AP1 protein were analyzed and predicted by using bioinformatics. Moreover, the differential expression of AP1 gene in the inner and outer petals among different development stages was systematically analyzed. Primers were designed based on the conserved sequence of higher plants AP1 gene and were used in amplifying the Paeonia lactiflora AP1 by RACE technology. Sequencing assays showed that the full-length cDNA of AP1 gene consisted of 1 236 bp and contained a 726 bp ORF encoding 242 amino acids. Bioinformatics analysis demonstrated that AP1 protein was unstable and hydrophilic, meanwhile no signal peptide and transmembrane structure indicating AP1 protein of non-secretory. α-helices and random coils were its main elements of the secondary structure of AP1 protein. AP1 protein had one N-glycosylation site located in 89th amino acid, one O-glycosylation site located in 206th amino acid, 13 potential phosphorylation sites including 8 conservative binding sites of specific protein kinase such as protein kinase C(PKC), epidermal growth factor receptor (EGFR), casein kinaseⅡ(CKⅡ), DNA-protein kinase (DNAPK), protein kinase A (PKA), protein kinase G (PKG), ataxia-telangiectasia mutated protein (ATM), p38-mitogen activated protein kinases (p38MAPK). AP1 protein had 2 super family conservative domains of MADS and K-box, which located in the region 2nd ~74th and 89th~173rd amino acid, respectively. Gene expression profile showed the expression level of AP1 gene was different in the inner and outer petals from a flower color chimaera cultivar Jinhui among 4 developmental stages (flower-bud stage, initiating bloom stage, bloom stage and wither stage). Especially, AP1 expressed very significantly different between the inner and outer petals in wither stage (P＜0.01), which furtherly indicated that the expression level of AP1 gene might be related to Paeonia lactiflora flower organ development. This study successfully obtained the full-length cDNA of AP1 gene and revealed the physicochemical property, structure and potential function of AP1 protein, which lays a foundation for further studying on the function of Paeonia lactiflora AP1.

The experiment was conducted to improve recombinant plectasin production, and evaluate its prospect in animal production. For optimal production of recombinant plectasin, the high-density fermentation of engineered Pichia pastoris (PPle) was performed in a 30-L fermenter. Through feed-batch fermentation process and glycerol basal salts medium, recombinant plectasin was prepared and its effects on immune function and intestinal health in rats (Rattus norvegicus) challenged with Staphylococcus aureus were investigated. After methanol induction at 30 ℃ for 72 h, the highest cell density and the maximal total protein concentration of the supernatant were achieved to 402 and 3.9 g/L, respectively. Animal experiment indicated that Staphylococcus aureus challenge did not affect the immune organ index and serum immune globulin (P＞0.05), but decreased duodenum and jejunum villus height and secretory immunoglobulin A (sIgA) concentration, however a higher villus height and secretory IgA in the small intestine were observed when injected with recombinant plectasin (P＜0.05). In addition, recombinant plectasin can decreased the total bacteria and Staphylococcus aureus of cecum (P＜0.05). The results indicate that the recombinant plectasin has a great potential in animal production, and it may be an ideal replacement for antibiotics in preventing and curing Staphylococcus aureus.

The high-affinity potassium ion transporter protein (HAK) plays an important role in improving the salt-tolerance in plants. In this study, we investigated the effect of salt stress on germination rate (Gr), germination potential (Gp), and germination index (Gi) in seeds of NtHAK1-overexpression plants and on the content of chlorophyll (Chl) and malondialdehyde (MDA), the activity of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) in NtHAK1-overexpressing tobacco plantlets (4 to 5 leaf stage). The results showed there was no siginificant effect on Gr, Gp, and Gi in the seeds of overexpressing and wild-type tobaccos under 50 mmol/L NaCl, but siginificant difference appeared between overexpressing plants and wild-type plants with 81.6% for Gr, 85.0% for Gp, and 42.6% for Gi in transgenic tobaccos under 100 mmol/L NaCl and 63.9% for Gr, 78.8% for Gp, and 20.2% for Gi in transgenic tobaccos under 150 mmol/L NaCl, which both were significantly higher than that in wild-type tobaccos (P＜0.05). The Chl content decreased from 2.25 mg/g (FW) before salt treatment to 1.31 mg/g(FW) at 5 d after salt treatment in wild-type plantlets and the decreasing amplitude of Chl content was 41.8%, while the average value of decreasing amplitude was 36.5% in transgenic tobaccos, which was significantly lower than the former. At 5 d after salt treatment, the MDA content was 105 nmol/g(FW) in non-transgenic plants, while it was 96.52 nmol/g(FW) in overexpressing plants. Meanwhile, the activities of antioxidant enzymes (AOEs), including SOD and CAT, were significantly up-regulated in NtHAK1-overexpression tobaccos with the increasing amplitude of 38.8% for SOD activity and 58.1% for CAT activity, while it was 34.2% for SOD and 54.6% for CAT in wild-type ones. The results from quantitative Real-time PCR for Na+/K+ absorption-related genes at 3 d after salt treatment in overexpressing and wild-type tobacco showed the relative expression levels of HAK1, potassium outward rectifier channel protein (TORK1), and vacuolar Na+/H+ antiporter protein (NHX1) were significantly up-regulated in NtHAK1- overexpression tobacco plants compared with wild-type ones. They were 3.85 folds for HAK1, 1.79 folds for TORK1, and 1.69 folds for NHX1 higher than those in wild-type tobaccos. In addition, the ratio of Na to K in different plant tissues demonstrated that it was 0.110 in root tissue and 0.106 in leaf tissue of transgenic plants respectively, while it was 0.147 for root tissue and 0.135 for leaf tissue in wild-type plants. The ratio of Na to K of different tissues in transgenic tobaccos was significantly lower (P＜0.05) than that in wild-type ones. This study provids the plant materials for breeding the salt-tolerant tobaccos and further studying the mechanism of HAK1-mediated salt-tolerance in transgenic plants.

Silkworm (Bombyx mori) 30K proteins are the major protein in the fifth instar larval hemolymph and possess multiple biological functions, such as inhibiting cell apoptosis induced by peroxide, virus or chemically and apolipoprotein functions. As a kind of insect apolipoprotein, they can bind with lipids and transport lipids inside insect larval hemolymph. Therefore it's very likely that silkworm 30K protein also has a similar function in mammalian cells, but it has not been reported so far. In order to study the effect of silkworm 30K apolipoprotein on lipid metabolism in mammalian cells, oleic acid (OA) and palmitic acid (PA) (OA∶PA=2∶1) solution was used to induce the normal human (Homo sapiens) liver cell line L-02 for 24 h to establish the fatty liver cell model in vitro. Then, the model was used to analyze the effects of these two kinds of silkworm 30K proteins, 30Kc6 and Lip-19G1, on lipid accumulation in liver cells. In order to understand the prevention function of these two proteins on lipid accumulation in liver cells, the normal liver cells were pretreated with 30Kc6 or Lip-19G1 protein before induction and then the cells were used to detect the lipid droplets IOD (integral optical density) value intracellular/total cell area by oil red O staining. The results showed that there was significant difference between 30Kc6 pretreatment group with control group (P＜0.05). However, there was no difference between Lip-19G1 pretreatment group with control groups. Morever, in order to understand the treatment effects of these two proteins on lipid accumulation in the fatty liver cells, the fatty liver cells were treated with 30Kc6 or Lip-19G1 protein, respectively. The fatty liver cells with 30Kc6 or Lip-19G1 protein treatment, the lipid droplets IOD value intracellular/ total cell area all reduced significantly compared with the cells without treatments (P＜0.01). These results showed that 30Kc6 had a certain inhibitory effects on the accumulation of lipid in human hepatic cells. Meanwhile, both 30Kc6 and Lip-19G1 proteins, could also reduce the accumulation of lipid droplet in fatty liver cells model. Therefore, this study will provide basic information for further study on the 30K apolipoprotein function and theoretical basis for new type of cholesterol drugs development.

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli results in serious damage on leaves and fruits in many cucurbit crops. To promote the biocontrol strategy of BFB, one Bacillus sp. strain TS86 was isolated from infected melon seeds with efficient biocontrol potential in previous study. In vitro dual-culture test showed that TS86 had strong antagonist inhibitory against to A. citrulli. It was also detected that TS86 was safe and effective to seeds germinating of muskmelon (Cucumis melo) and water melon (Citrullus lanatus). Furthermore, TS86 was used to prepare biological seed coating agent by using its fermentation liquor as active ingredient, in order to accelerate the TS86 related product development and application. Seed coating agents screen test displayed 4% polyvinyl alcohol AH-26 was selected as the optimal film former for good film forming ability, water resistant property, water permeability, air permeability and swelling capacity, with no harm to biocontrol bacteria; 5% ethylene plycol was selected as the optimal antifreeze for its acceptable property of resistance to frost; 0.3% fuchsin acid was selected as the optimal warning coloration for it had batter boating color. In addition, 5% sucrose was added as the best combination. Consequently, biological seed coating agent was prepared by mixing the fermented liquid of biocontrol bacteria TS86 and various auxiliaries with a certain ratio. The determination of the indicators revealed the initial concentration of TS86 in seed coating agent was 4.0×108~5.0×108 CFU/mL, with weak acidity (pH 5.70) and the viscosity was 46 mPa/s. The bacteria concentration was 5.1×106 CFU/mL after storage for 1 year which suggested that TS86 could maintain high level concentration in room temperature for a long time. Greenhouse tests were carried out to evaluate the biocontrol efficacy of TS86, in which plant seeds with pathogen were pretreated with seeds coating at the dosage of 1∶50 and biocontrol effectiveness of the biological seed agent. The results indicated that, the biocontrol efficacy against BFB in watermelon and muskmelon were increased by 70.63% and 80.59%, respectively. All together, we could conclude that the biological coating agent which was created by TS86 was highly effective in BFB controlling during melon production. To the best of our knowledge, its characteristics of high efficiency, safety and environmental protection has a great significance in sustainable control of BFB and reduce of pesticide pollution.

The selection of a suitable reference gene is an important prerequisite for successful gene expression analysis by quantitative Real-time PCR (qRT-PCR). In this study, expression stability of 22 endogenous candidate genes including 16 new candidate reference genes form transcriptome data of tree peony, including AMPDS, ARFA1C, ERVTP, FCF2PRP, GATA24, GRF9, MBF1A, MHCRP, PAD1FP, PIN1AT, PKSP, PP2A, PP2CFP, PTRP, PUF1639, RPS9 and 6 traditional housekeeping genes, including ARP2, IWS1, GAPDH, TBCC, TUA5 and UBC28 were assessed by qRT-PCR in a diverse set of 33 tissue samples representing different floral development stages of ‘Feng Dan’(Paeonia ostii ‘Feng Dan’), ‘Xi Shi’(Paeonia ostii ‘Xi Shi’) and ‘Que Hao’(Paeonia ostii ‘Que Hao’), different color of ‘Feng Dan’, different samples (stems, leaves, sepals and flowers), different development stages of seeds. The results showed that the most suitable candidate reference genes of tree peony had many differences in the various experimental condition groups. Though there were some minor differences, the results of two programs were similar in the same experimental condition group. ERVTP, PP2CFP, FCF2PRP and UBC28 had the highest expression stability in flowers of different floral development stages in Feng Dan, Xi Shi and Que Hao. RPS9 and ARFA1C were the best genes in flowers of different color of Feng Dan. AMPDS, PUF1639 and MBF1A were the most stable reference genes which were suitable for different samples (stems, leaves, sepals, petals). In seeds of different development stages, RPS9 and PUF1639 had the stable expression. Further, four newly selected reference genes (PUF1639, MBF1A, PP2CFP and RPS9) of tree peony were superior to traditional ones in terms of their expression stability. Identifying stably expressed genes for candidate reference genes from transcriptome database was found to be reliable and efficient. The reference genes selected in this study will be helpful to improve the quality of gene expression studies in tree peony.

In order to establish and optimize the two-dimensional electrophoresis (2-DE) system for testis proteins of matured male sheep (Ovis aries), different extracting methods, loading quantities, isoelectric focusing time and separation gel concentration were compared of their influence on the 2-DE maps. Results indicated that the best 2-DE maps were obtained through preparing proteins samples by the trichloroacetic acid(TCA)/acetone precipitation, loading 750 μg protein samples, isoelectric focusing with 75 000 Vh or more and 12% sodium dodecyl sulfonate (SDS) gel for protein separation. The optimized 2-DE system gives the maximum number of protein spots with clear background, which help to the study of male ovine testis development and male reproductive disease.